The present exemplary embodiments relate to the imaging arts, and find particular application in conjunction with low and high-density cell detection, locating, and identifying in blood smears, biological assays, and the like across distinct imaging systems, and will be described with particular reference thereto. However, it is to be appreciated the exemplary embodiments will also find application in imaging, locating and identifying other types of low- or high-density features on various substantially planar surfaces and samples, such as imaging semiconductor wafers, imaging particulate contaminants in fluids or thin solid films, and so forth, with such imaging finding specific uses in the printing arts, electronic arts, medical arts, and other scientific and engineering areas.
In rare cell studies, a particular problem arises due to the typically low concentration of the rare cells in the blood or other body fluid. In a typical rare cell study, blood is processed to remove cells that that are not needed. Then a fluorescent material is applied that attaches to antibodies, which in turn selectively attach to a cell surface or cellular protein of the rare cells. The cellular proteins may be membrane proteins or proteins within a cell, such as cytoplasm proteins. The antibodies may also attach to other types of molecules of the rare cell, as well as to DNA.
The fluorescent material may be a fluorescent marker dye or any other suitable material which will identify the cells of interest. A smear treated in this manner, which may include the blood and/or components of the blood, is prepared and optically analyzed to identify rare cells of the targeted type. For statistical accuracy it is important to obtain as large a number of cells as required for a particular process, in some studies at least ten rare cells should be identified, requiring a sampling of at least ten million cells, for a one-in-one-million rare cell concentration. Such a blood smear typically occupies an area of about 100 cm2. It is to be understood, however, that this is simply one example and other numbers of cells may be required for statistical accuracy for a particular test or study. Other cell identifiers which are being used and investigated are quantum dots and nano-particle probes. Also, while a rare cell is mentioned as a one-in-one-million cell concentration, this is not intended to be limiting and is only given as an example of the rarity of the cells being sought. The concepts discussed herein are to be understood to be useful in higher or lower levels of cell concentration.
In this regard, the ability to scan large numbers of cells at a high rate is considered a key aspect which increases the throughput of testing processes. Therefore, it is considered valuable to provide a system which improves the speed, reliability and processing costs which may be achieved by cell detection systems and/or processes.
A number of cell detection techniques have been proposed including fluorescence in situ hybridization (FISH), flow cytometry, laser scanning cytometry (LSC), among others.
While the above-noted systems are directed to creating faster scan rates, they nevertheless still have relatively small fields of view (FOV), such as microscopes. This will, therefore, still result in speeds which do not reach the desired scan rates.
In view of this, the previously noted and incorporated U.S. application Ser. No. 10/271,347 discloses a fiber array scanning technology (FAST) that increases the speed at which scanning of a sample and the detection of potential or candidate rare cells may be accomplished, lending itself to the investigation of large samples. Still, while the aforementioned application provided an increased speed, a still further increase in speed can be accomplished by, e.g., providing a second laser that produces excitation light at a second wavelength or wavelength range and a second signal detector calibrated to sense a second fluorescence signal simultaneously with the first laser and signal detector. In this arrangement, each signal detector could be configured with a filter to sense only the desired respective fluorescence signal. This essentially doubles the amount of information that can be detected during a single scan.
One problem that arises with this arrangement, however, is that a portion of either stimulated fluorescence signal may significantly overlap the remaining fluorescence signal in terms of wavelength. Selective filtering can reduce this problem but at the cost of reducing the useful wavelength band that may be sensed. A second problem that arises is that this method would normally make use of a beam splitter or dichroic mirror in the light path of the fluorescent radiation in order to direct desired portions of the fluorescent signal to the respective signal detector, further reducing the intensity of the signal being sensed.